Highly Hydrophilic Segments Attached to Hydrophobic Peptides Translocate Rapidly across Membranes

Hydrophilic segments attached to transmembrane helices often cross membranes. In an increasing number of cases, it has become apparent that this occurs in a biologically relevant post-translational event. In this study, we investigate whether juxta-membrane (JM) hydrophilic sequences attached to hydrophobic helices are able to rapidly cross lipid bilayers via their ability or inability to block hydrophobic helix interconversion between a transmembrane (TM) and non-TM membrane-associated state. Interconversion was triggered by changing the protonation state of an Asp residue in the hydrophobic core of the peptides, and peptide configuration was monitored by the fluorescence of a Trp residue at the center of the hydrophobic sequence. In POPC vesicles, conversion of the TM to non-TM state at high pH and the non-TM to TM state at low pH was rapid (seconds or less) for KK, KKNN, and the KKNNNNNN flanking sequences on both N- and C-termini and the KLFAGHQ sequence that flanks the spontaneously TM-inserting 3A protein of polio virus. In vesicles composed of 6:4 (mol/mol) POPC/cholesterol, interconversion was still rapid, with the exception of the peptide flanked by KKNNNNNN sequences, for which the half time of interconversion slowed to minutes. This behavior suggests that, at least in membranes with low levels of cholesterol, movement of hydrophilic JM segments (and analogous hydrophobic loops in multipass TM proteins) across membranes may be more facile than previously thought. This may have important biological implications.